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肽抗生素hPAB-β制备工艺中基因工程菌的改建 被引量:1

Reconstruction of engineered bacteria in preparing recombinant human peptide antibiotic β
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摘要 目的通过重新构建新的3拷贝串联体基因工程菌phPAB-β(3)/M15,在原有肽抗生素hPAB-β制备工艺路线基础上,使发酵后目的肽的最终产量得到提高。方法重新构建含有重组质粒pQE31-hPAB-β(3)的基因工程菌M15,然后筛选出能够稳定而且高效表达目标蛋白的最佳工程菌株,进行传代稳定性分析,并明确新建工程菌中目标融合蛋白的表达形式和亲和层析的效果,最后通过相同条件下,在逐步放大的发酵规模(1L摇瓶,3.7L和10L发酵罐),与以往的3拷贝串联体工程菌phPAB-β(3)/JM109进行对比研究(湿菌产量、目标蛋白表达率),同时观察新建工程菌在10L发酵中目标蛋白的表达和遗传稳定性。结果筛选出的最佳工程菌株的目标蛋白的表达效率为34.8%,经LB培养传代10次后,点种LB(AMP、KAN)平板,生长率为100%,质粒保存率达100%。在1L摇瓶,3.7L和10L发酵罐的发酵规模下,phPAB-β(3)/M15与phPAB-β(3)/JM109一样,目标融合蛋白都以包涵体形式表达,亲和层析效果相近,目标蛋白表达率无显著性差异(P>0·05),但发酵产量大于后者,有非常显著性差异(P<0·01)。且新建工程菌在10L发酵中发酵产量达到55.15g/L,诱导表达后的第5小时,蛋白表达达到高峰,蛋白表达率为30.2%,重组质粒保存率为100%。结论新构建工程菌phPAB-β(3)/M15比起原来基因工程菌phPAB-β(3)/JM109,是更为理想且利于肽抗生素hPAB-β工业生产的基因工程菌。 Objective To reconstruct the new engineered bacteria expressing hPAB-β triploids so as to improve the outputs of recombinant human peptide antibiotic β. Methods The recombinant plasmid pQE31- hPAB-β(3) was transformed into E. coll. M15 to screen the new engineered bacteria expressing hPAB-β triploids. The stabilities of phPAB-β(3)/M15 were observed in continuous cultures. The expression levels of the fusion peptides of interest and the bacterial yields of the new engineered bacteria phPAB-β(3)/M15 were compared with that ofphPAB-β(3)/JM109 in different fermentation scales. Results Genetic stability of the recombinant plasmid and phPAB-β(3)/M15 was 100% after 10 passages. Take bacterial yields into account, the new engineered bacteria phPAB-β ( 3 )/M15 was better than phPAB-β ( 3 )/JM109 at the similar expression levels of the target proteins by "t" test analysis (P 〈0.01 ). The yields of βrmentation reached 55.15 g/L in 10 L fermenter, and the expression levels were up to 30.2% at 5th h after induction. During the fermentation, the recombinant plasmids were stable in culture. Conclusion The new engineered bacterium phPAB-β(3)/M15 successfully constructed was more consummate and effective on the basis of the original process.
出处 《第三军医大学学报》 CAS CSCD 北大核心 2006年第22期2243-2246,共4页 Journal of Third Military Medical University
基金 国家自然科学基金资助项目(30171119) 全军医学科学技术研究"十五"计划基金重点课题(01Z070) 国家"863"项目(2002AA214211)~~
关键词 肽抗生素 hPAB—β 发酵 M15 peptide antibiotics hPAB-β fermentation M15
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参考文献7

  • 1REDDY Q V,YEDERY R D,ARANHA C.Antimicrobial peptides:premises and promises[J].Int J Antimicrob Agents,2004,24 (6):536-547.
  • 2胡金川,饶贤才,黎庶,金晓琳,汪正清,胡福泉.人肽抗生素hPABβ重组质粒的构建及其在大肠杆菌中的表达[J].解放军医学杂志,2004,29(2):113-115. 被引量:5
  • 3孟洁如,颜真,赵宁,张英起.导向性人干扰素α2a工程菌的高密度发酵[J].第四军医大学学报,2004,25(23):2144-2147. 被引量:10
  • 4AKIYAMA N,HIJIKATA M,KOBAYASHI A,et al.Anti-tumor effect of N-beta-alanyl-5-S-glutathionyldihydroxyphenylalanine (5-S-GAD),a novel anti-bacterial substance from an insect[J].Anticancer Res,2000,20(1 A):357-362.
  • 5LEEM J Y,JEONG I J,PARK K T,et al.Isolation of p-hydroxycinnamaldehyde as an antibacterial substance from the saw fly,Acantholyda parki S[J].FEBS Lett,1999,442(1):53 -56.
  • 6QIAGEN Companies.The QIA expressionistTM,Fifth Edition,2003,6.
  • 7GOTTESMAN S,HALPERN E,TRISLER P.Role of sulA and sulB in filamentation by lon mutants of Escherichia coli K-12[J].J Bacteriol,1981,148(1):265 -273.

二级参考文献12

  • 1Eric J, Frank GH. Interferon in oncological practice: Review of interferon biology, clinical applications, and toxicities [J]. Oncology, 2001; 6: 34-55.
  • 2Pfeffer LM, Dinarello CA, Herberman RB, et al. Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons [J]. Cancer Res, 1998; 58: 2489-2499.
  • 3Wadih A, Renata P, Erkki R. Cancer treatment by targeted drug delivery to tumor vasculature in mouse model[J]. Science, 1998; 279(16): 377-380.
  • 4Makides SC. Strategies for the achieving high-level expression of genes in Escherichia coli[J]. Microbiol Rev, 1996; 60(3): 512-538.
  • 5Khalilzadeh R, Shojaosadati SA, Bahrami A, et al. Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli[J]. Biotechnol Lett, 2003;25(23):1989-1992.
  • 6Tabandeh F, Shojaosadati SA, Zomorodipour A, et al. Heat-induced production of human growth hormone by high cell density cultivation of recombinant Escherichia coli[J]. Biotechnol Lett, 2004;26(3):245-250.
  • 7Srinivasan S, Barnard GC, Gerngross TU. Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha[J]. Biotechnol Bioeng, 2003;84(1):114-120.
  • 8Baheri HR, Roesler WJ, Hill GA. Modeling of recombinant bacteria fermentation for enhanced productivity[J]. Biotechnol Techniq, 1997; 17(2): 243-251.
  • 9Panda AK. Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli[J]. Adv Biochem Eng Biotechnol, 2003;85:43-93.
  • 10饶贤才,胡晓梅,张克斌,金晓琳,朱军民,刘启富,张光明,胡福泉.人源肽抗生素hBD-2表达质粒的构建及其在大肠杆菌中的表达[J].微生物学免疫学进展,2001,29(3):1-5. 被引量:3

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